GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue.

The [S]GTPγS assay is a method that measures the level of G protein activation by determining the binding of [S]GTPγS, a non-hydrolyzable and radioactively labeled GTP analog, to Gα subunit of heterotrimeric G protein upon activation of G protein-coupled receptors (GPCR). The power of this assay lies in the fact that it measures an early event of GPCR signaling in cells expressing recombinant receptors and cells and tissues expressing endogenous receptors. The present protocol describes a sensitive method for studying G protein activation by melatonin receptors MT and MT, in membranes prepared from mouse brain.

Apelin and Vasopressin: The Yin and Yang of Water Balance.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling body fluid homeostasis and cardiovascular functions. Experimental data performed in rodents have shown that apelin has an aquaretic effect its central and renal actions. In the brain, apelin inhibits the phasic electrical activity of vasopressinergic neurons and the release of vasopressin from the posterior pituitary into the bloodstream and in the kidney, apelin regulates renal microcirculation and counteracts in the collecting duct, the antidiuretic effect of vasopressin occurring the vasopressin receptor type 2.

Brain ACE2 activation following brain aminopeptidase A blockade by firibastat in salt-dependent hypertension.

In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation.

Pharmacological evidence for transactivation within melatonin MT and serotonin 5-HT receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of G -coupled melatonin MT receptors and G -coupled serotonin 5-HT receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT . Here, we identified this functional fingerprint in the mouse brain.

LGR5 controls extracellular matrix production by stem cells in the developing intestine.

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage.

Melatonin MT and MT receptor ERK signaling is differentially dependent on G and G proteins.

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT and MT receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules.

O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors.

A series of dimeric melatonin analogues 3a-e obtained by connecting two melatonin molecules through the methoxy oxygen atoms with spacers spanning 16-24 atoms and the agomelatine dimer 7 were synthesized and characterized in 2-[-I]-iodomelatonin binding assays, bioluminescence resonance energy transfer (BRET) experiments, and in functional cAMP and β-arrestin recruitment assays at MT and MT receptors. The binding affinity of 3a-e generally increased with increasing linker length. Bivalent ligands 3a-e increased BRET signals of MT dimers up to 3-fold compared to the monomeric control ligand indicating the simultaneous binding of the two pharmacophores to dimeric receptors.

Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT receptor reciprocally modulate their signaling functions.

Understanding the function of orphan G protein-coupled receptors (GPCRs), whose cognate ligand is unknown, is of major importance as GPCRs are privileged drug targets for many diseases. Recent phylogenetic studies classified three orphan receptors, GPR61, GPR62 and GPR135 among the melatonin receptor subfamily, but their capacity to bind melatonin and their biochemical functions are not well characterized yet. We show here that GPR61, GPR62 and GPR135 do not bind [H]-melatonin nor 2-[I]iodomelatonin and do not respond to melatonin in several signaling assays.

Design and validation of the first cell-impermeant melatonin receptor agonist.

Background And Purpose: The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT and MT receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools.

Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F.

New structural insights into the apelin receptor: identification of key residues for apelin binding.

Apelin is the endogenous ligand of the orphan 7-transmembrane domain GPCR APJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3-dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor-1 3D model or the X-ray structures of the β2-adrenergic and CXCR4 receptors as templates.

By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor.